Improving the Developability of an Antigen Binding
Fragment by Aspartate Substitutions
Version 2 2019-06-06, 11:47Version 2 2019-06-06, 11:47
Version 1 2019-06-05, 14:33Version 1 2019-06-05, 14:33
Posted on 2019-06-06 - 11:47
Aggregation
can be a major challenge in the development of antibody-based
pharmaceuticals as it can compromise the quality of the product during
bioprocessing, formulation, and drug administration. To avoid aggregation,
developability assessment is often run in parallel with functional
optimization in the early screening phases to flag and deselect problematic
molecules. As developability assessment can be demanding with regard
to time and resources, there is a high focus on the development of
molecule design strategies for engineering molecules with a high developability
potential. Previously, Dudgeon et al. [(2012) Proc. Natl.
Acad. Sci. U. S. A. 109, 10879–10884] demonstrated
how Asp substitutions at specific positions in human variable domains
and single-chain variable fragments could decrease the aggregation
propensity. Here, we have investigated whether these Asp substitutions
would improve the developability potential of a murine antigen binding
fragment (Fab). A full combinatorial library consisting of 393 Fab
variants with single, double, and triple Asp substitutions was first
screened in silico with Rosetta; thereafter, 26 variants
with the highest predicted thermodynamic stability were selected for
production. All variants were subjected to a set of developability
studies. Interestingly, most variants had thermodynamic stability
on par with or improved relative to that of the wild type. Twenty-five
of the variants exhibited improved nonspecificity. Half of the variants
exhibited improved aggregation resistance. Strikingly, while we observed
remarkable improvement in the developability potential, the Asp substitutions
had no substantial effect on the antigenic binding affinity. Altogether,
by combining the insertion of negative charges and the in
silico screen based on computational models, we were able
to improve the developability of the Fab rapidly.
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Sakhnini, Laila I.; Greisen, Per J.; Wiberg, Charlotte; Bozoky, Zoltan; Lund, Søren; Wolf Perez, Adriana-Michelle; et al. (2019). Improving the Developability of an Antigen Binding
Fragment by Aspartate Substitutions. ACS Publications. Collection. https://doi.org/10.1021/acs.biochem.9b00251